Embodiments of the present invention generally broadly relate to living mammalian cells. More specifically, embodiments of the present invention generally provide for the preservation and survival of human cells, especially eukaryotic cells and erythrocytic cells.
Embodiments of the present invention also generally broadly relate to the therapeutic uses of blood platelets, eukaryotic cells, and erythrocytic cells; and more particularly to manipulations or modifications of platelets, eukaryotic cells, and erythrocytic cells, such as in preparing freeze-dried compositions that can be rehydrated at the time of application. When freeze-dried platelets are rehydrated, they have a normal response to thrombin and other agonists with respect to that of fresh platelets. When eukaryotic cells and erythrocytic cells are rehydrated, they are immediately restored to viability.
The inventive compositions and methods for embodiments of the present invention are useful in many applications, such as in medicine, pharmaceuticals, biotechnology, and agriculture, and including transfusion therapy, as hemostasis aids and for drug delivery.
Blood transfusion centers are under considerable pressure to produce platelet concentrates for transfusion. The enormous quest for platelets necessitates storage of this blood component, since platelets are important contributors to hemostasis. Platelets are generally oval to spherical in shape and have a diameter of 2-4 xcexcm. Today platelet rich plasma concentrates are stored in bloodbags at 22xc2x0 C.; however, the shelf life under these conditions is limited to five days. The rapid loss of platelet function during storage and risk of bacterial contamination complicates distribution and availability of platelet concentrates. Platelets tend to become activated at low temperatures. When activated they are substantially useless for an application such as transfusion therapy. Therefore the development of preservation methods that will increase platelet lifespan is desirable.
Several techniques for preservation of platelets have been developed over the past few decades. Cryopreservation of platelets using various agents, such as glycerol (Valeri et al., Blood, 43, 131-136, 1974) or dimethyl sulfoxide, xe2x80x9cDMSOxe2x80x9d (Bock et al., Transfusion, 35, 921-924, 1995), as the cryoprotectant have been done with some success. The best results have been obtained with DMSO. However, a considerable fraction of these cells are partly lysed after thawing and have the shape of a balloon. These balloon cells are not responsive to various agonists, so that overall responsiveness of frozen thawed platelets to various agonists is reduced to less than 35% compared with fresh platelets. The shelf life of cryopreserved DMSO platelets at xe2x88x9280xc2x0 C. is reported to be one year, but requires extensive washing and processing to remove cryoprotective agents, and even then the final product has a severe reduction in ability to form a clot.
Attempts to dry platelets by lyophilization have been described with paraformaldehyde fixed platelets (Read et al., Proc. Natl. Acad. Sci. USA, 92, 397401, 1995). U.S. Pat. No. 5,902,608, issued May 11, 1999, inventors Read et al. describe and claim a surgical aid comprising a substrate on which fixed, dried blood platelets are carried. These dried blood platelets are fixed by contacting the platelets to a fixative such as formaldehyde, paraformaldehyde, gutaraldehyde, or permanganate. Proper functioning of lyophilized platelets that have been fixed by such fixative agents in hemostasis is questionable.
Spargo et al., U.S. Pat. No. 5,736,313, issued Apr. 7, 1998, have described a method in which platelets are loaded overnight with an agent, preferably glucose, and subsequently lyophilized. The platelets are preincubated in a preincubation buffer and then are loaded with carbohydrate, preferably glucose, having a concentration in the range of about 100 mM to about 1.5 M. The incubation is taught to be conducted at about 10xc2x0 C. to about 37xc2x0 C., most preferably about 25xc2x0 C.
U.S. Pat. No. 5,827,741, Beattie et al., issued Oct. 27, 1998, discloses cryoprotectants for human platelets, such as dimethylsulfoxide and trehalose. The platelets may be suspended, for example, in a solution containing a cryoprotectant at a temperature of about 22xc2x0 C. and then cooled to below 15xc2x0 C. This incorporates some cryoprotectant into the cells.
Trehalose is a disaccharide found at high concentrations in a wide variety of organisms that are capable of surviving almost complete dehydration (Crowe et al., Anhydrobiosis. Annul. Rev. Physiol., 54, 579-599, 1992). Trehalose has been shown to stabilize certain cells during freezing and drying (Leslie et al., Biochim. Biophys. Acta, 1192, 7-13, 1994; Beattie et al., Diabetes, 46, 519-523, 1997).
Other workers have sought to load platelets with trehalose through use of electroporation before drying under vacuum. However, electroporation is very damaging to the cell membranes and is believed to activate the platelets. Activated platelets have dubious clinical value.
Platelets have also been suggested for drug delivery applications in the treatment of various diseases, as is discussed by U.S. Pat. No. 5,759,542, issued Jun. 2, 1998, inventor Gurewich. This patent discloses the preparation of a complex formed from a fusion drug including an A-chain of a urokinase-type plasminogen activator that is bound to an outer membrane of a platelet.
Accordingly, a need exists for the effective and efficient preservation of platelets such that they maintain, or preserve, their biological properties, particularly their response to platelet agonists such as thrombin, and which can be practiced on a large, commercially feasible scale. Further, it would also be useful to expand the types of present vehicles that are useful for encapsulating drugs and used for drug delivery to targeted sites. Accordingly further, a need also exists for the effective and efficient preservation of eukaryotic cells and erythrocytic cells, such that these cells respectively maintain their biological properties and may readily become viable after storage.
In one aspect of the present invention, a dehydrated composition is provided comprising freeze-dried platelets that are effectively loaded with trehalose to preserve biological properties during freeze-drying and rehydration. These platelets are rehydratable so as to have a normal response to at least one agonist, such as thrombin. For example, substantially all freeze-dried platelets of the invention when rehydrated and mixed with thrombin (1 U/ml) form a clot within three minutes at 37xc2x0 C. The dehydrated composition can include one or more other agents, such as antibiotics, antifungals, growth factors, or the like, depending upon the desired therapeutic application.
In another aspect of the present invention, a hemostasis aid is provided where the above-described freeze-dried platelets are carried on or by a biocompatible surface. A further component of the hemostasis aid may be a therapeutic agent, such as an antibiotic, an antifungal, or a growth factor. The biocompatible surface may be a bandage or a thrombic surface, such as freeze-dried collagen. Such a hemostasis aid can be rehydrated just before the time of application, such as by hydrating the surface on or by which the platelets are carried, or, in case of an emergency, the dry hemostasis treatment aid could be applied directly to the wound or burn and hydrated in situ.
Methods of making and using inventive embodiments are also described. One such method is a process of preparing a dehydrated composition comprising providing a source of platelets, effectively loading the platelets with trehalose to preserve biological properties, cooling the trehalose loaded platelets to below their freezing point, and lyophilizing the cooled platelets. The trehalose loading includes incubating the platelets at a temperature from greater than about 25xc2x0 C. to less than about 40xc2x0 C. with a trehalose solution having up to about 50 mm trehalose therein. The process of using such a dehydrated composition further may comprise rehydrating the platelets. The rehydration preferably includes a prehydration step wherein the freeze-dried platelets are exposed to warm, saturated air for a time sufficient to bring the water content of the freeze-dried platelets to between about 20 weight percent to about 35 weight percent.
In yet another aspect of the present invention, a drug delivery composition is provided comprising platelets having a homogeneously distributed concentration of a therapeutic agent therein. The drug delivery composition is particularly useful for targeting the encapsulated drug to platelet-mediated sites.
Practice of the present invention permits the manipulation or modification of platelets while maintaining, or preserving, biological properties, such as a response to thrombin. Further, use of the method to preserve platelets can be practiced on a large, commercially feasible scale, and avoids platelet activation. The inventive freeze-dried platelets, and hemostasis aids including the freeze-dried platelets, are substantially shelf stable at ambient temperatures when packaged in moisture barrier materials.
Embodiments of the present invention also provide a process for preserving and/or increasing the survival of dehydrated eukaryotic cells after storage comprising providing eukaryotic cells from a mammalian species (e.g., a human); loading the eukaryotic cells with a preservative (e.g., an oligosaccharide, such as trehalose); dehydrating the eukaryotic cells while maintaining a residual water content in the eukaryotic cells greater than about 0.15 (e.g., from about 0.20 to about 0.75) gram of water per gram of dry weight eukaryotic cells to increase eukaryotic cell survival, preferably to greater than about 80%, upon rehydrating after storage; storing the dehydrated eukaryotic cells having the residual water content greater than about 0.15 gram of water per gram of dry weight eukaryotic cells; and rehydrating the stored dehydrated eukaryotic cells with the stored dehydrated eukaryotic cells having an increase in survival following dehydration and storage. In a preferred embodiment, more than about 80% of the stored dehydrated cells survive the dehydration and storage.
Embodiments of the present invention further provide a process of preparing loaded eukaryotic cells comprising providing eukaryotic cells selected from a mammalian species; and loading (e.g., with an oligosaccharide solution and/or with or without a fixative) an oligosaccharide (e.g., trehalose) into the eukaryotic cells at a temperature greater than about 25xc2x0 C. (e.g., greater than about 25xc2x0 C. but less than about 50xc2x0 C., such as from about 30xc2x0 C. to less than about 50xc2x0 C., or from about 30xc2x0 C. to about 40xc2x0 C.) to produce loaded eukaryotic cells. The loading comprises taking up external oligosaccharide via fluid phase endocytosis from an oligosaccharide solution at the temperature greater than about 25xc2x0 C. The loading further comprises incubating the eukaryotic cells at the temperature greater than about 25xc2x0 C. with the oligosaccharide solution. For these embodiments of the present invention, the eukaryotic cells are preferably human eukaryotic cells, such as, by way of example only, eukaryotic cells selected from the group of eukaryotic cells consisting of mesenchymal stem cells and epithelial 293H cells.
Embodiments of the present invention also further provide a solution for loading eukaryotic cells comprising eukaryotic cells selected from a mammalian species; and an oligosaccharide solution containing the eukaryotic cells and a temperature greater than about 25xc2x0 C. for loading oligosaccharide from the oligosaccharide solution into the eukaryotic cells. External oligosaccharide is taken up via fluid phase endocytosis from the oligosaccharide solution at a temperature ranging from about 30xc2x0 C. to less than about 42xc2x0 C. An eukaryotic cell composition is also provided as broadly comprising eukaryotic cells loaded internally with an oligosaccharide, preferably trehalose, from an oligosaccharide solution at a temperature greater than about 25xc2x0 C.
Embodiments of the present invention yet also further provide a generally dehydrated composition comprising freeze-dried eukaryotic cells selected from a mammalian species (e.g., a human) and being effectively loaded internally (e.g., incubating the eukaryotic cells at a temperature from about 30xc2x0 C. to less than about 50xc2x0 C. so as to uptake external trehalose via fluid phase endocytosis) with at least about 10 mM trehalose therein to preserve biological properties during freeze-drying and rehydration. The amount of trehalose loaded inside the freeze-dried eukaryotic cells is preferably from about 10 mM to about 50 mM. The freeze-dried eukaryotic cells comprise at least about 0.15 (e.g., from about 0.20 to about 0.75) gram of residual water per gram of dry weight eukaryotic cells to increase eukaryotic cell survival upon rehydrating.
Aspects of embodiments of the present invention also include a process for preparing a dehydrated composition. The process comprises providing eukaryotic cells selected from a mammalian species (e.g., a human); loading internally the eukaryotic cells with from about 10 mM to about 50 mM of an oligosaccharide (e.g., trehalose) therein to preserve biological properties. The loading includes incubating the eukaryotic cells at a temperature from about 30xc2x0 C. to less than about 50xc2x0 C., preferably from about 30xc2x0 C. to about 40xc2x0 C., more preferably from about 34xc2x0 C. to about 37xc2x0 C., with an oligosaccharide solution having up to about 50 mM oligosaccharide therein; cooling the loaded eukaryotic cells to below their freezing point; and lyophilizing the cooled eukaryotic cells. Lyophilizing preferably is conducted so as to leave a residual water content of less than about 0.40 gram of water per gram of dry weight eukaryotic cells, preferably greater than about 0.15 gram of water per gram of dry weight eukaryotic cells, but more preferably less than about 0.40 gram of water per gram of dry weight of eukaryotic cells.
Further aspects of embodiments of the present invention include a process for increasing the loading efficiency of an oligosaccharide into eukaryotic cells. The process comprises providing eukaryotic cells having a first phase transition temperature range and a second phase transition temperature range (e.g., a temperature greater than about 25xc2x0 C., such as from about 30xc2x0 C. to less than about 50xc2x0 C.) which is greater than the first phase transition temperature range; disposing the eukaryotic cells in an oligosaccharide solution for loading an oligosaccharide (e.g., trehalose) into the eukaryotic cells; and heating the oligosaccharide solution to the second phase transition temperature range to increase the loading efficiency of the oligosaccharide into the eukaryotic cells. The process additionally comprises taking up external oligosaccharide via fluid phase endocytosis from the oligosaccharide solution.
The present invention also comprises additional embodiments which include a process for increasing the cooperativity of a phase transition of an erythrocytic cell comprising providing an erythrocytic cell having an alcohol (e.g. a sterol) and a phase transition, and removing at least a portion of the alcohol from the erythrocytic cell to increase the cooperativity of the phase transition of the erythrocytic cell. The erythrocytic cell preferably comprises an erythrocytic membrane including the alcohol and the phase transition. Another embodiment of the present invention provides a process for producing a phase transition temperature range in an erythrocytic cell comprising providing an erythrocytic cell including an alcohol and at least two phase transition temperature ranges, and removing at least a portion of the alcohol from the erythrocytic cell to produce an erythrocytic cell having at least three phase transition temperature ranges. The erythrocytic cell for this feature or aspect of the invention preferably includes an erythrocytic membrane including at least a portion of the alcohol and at least a portion of the two phase transition temperature ranges. After the erythrocytic cell is produced, the produced erythrocytic cell preferably comprises the erythrocytic membrane including at least a portion of the three phase transition temperature ranges after removal of at least a portion of the alcohol.
A further embodiment of the present invention provides a process for loading an oligosaccharide into erythrocytic cells comprising providing erythrocytic cells having an alcohol (e.g. a sterol); removing at least a portion of the alcohol from the erythrocytic cells to produce erythrocytic cells having a phase transition temperature range selected from the group of temperature ranges consisting of a low phase transition temperature range, an intermediate phase transition temperature range, and a high phase transition temperature range; and disposing the erythrocytic cells in an oligosaccharide solution for loading an oligosaccharide (e.g., trehalose) into the erythrocytic cells. The oligosaccharide solution preferably includes a temperature in a range that approximates the range of temperatures for the phase transition temperature range. The process for loading the oligosaccharide into the erythrocytic cells may additionally comprise heating the oligosaccharide solution, such as to a temperature in the high phase transition temperature range, to increase the loading efficiency of the oligosaccharide into the erythrocytic cells. The process may further additionally comprise taking up external oligosaccharide via lipid phase endocytosis from the oligosaccharide solution. The erythrocytic cells do not necessarily include a fixative.
Another embodiment of the present invention provides a process for increasing the survival of dehydrated erythrocytic cells after storage. The process for increasing survival preferably comprises providing erythrocytic cells from a mammalian species (e.g., a human being) and having an alcohol (e.g. a sterol); removing, preferably at least part of, the alcohol from the erythrocytic cells; and loading the erythrocytic cells with a preservative (e.g., an oligosaccharide). The loaded erythrocytic cells are then dehydrated (e.g., by lyophilizing) while maintaining a residual water content in the erythrocytic cells equal to or less than about 0.30 gram of residual water per gram of dry weight erythrocytic cells to increase erythrocytic cell survival upon rehydrating after storage. The process for increasing survival also preferably comprises storing the dehydrated erythrocytic cells having the residual water content equal to or less than about 0.30 gram of residual water per gram of dry weight erythrocytic cells; and rehydrating the stored dehydrated erythrocytic cells with the stored dehydrated erythrocytic cells surviving dehydration and storage. The loading may be without a fixative and may comprise taking up external oligosaccharide via lipid phase endocytosis from the oligosaccharide solution. The loading may also, or alternatively, comprise incubating the erythrocytic cells with the oligosaccharide solution. The loaded erythrocytic cells may be cooled to a temperature below their freezing point prior to dehydrating the erythrocytic cells. The residual water content of the erythrocytic cells preferably ranges from about 0.00 gram of residual water per gram of dry weight erythrocytic cells to less than about 0.30 gram of residual water per gram of dry weight erythrocytic cells.
A further embodiment of the present invention provides a process of preparing a dehydrated composition comprising providing erythrocytic cells selected from a mammalian species and including an alcohol (e.g. a sterol); loading internally the erythrocytic cells with more than about 10 mM of an oligosaccharide therein to preserve biological properties; cooling the loaded erythrocytic cells to below their freezing point; and lyophilizing the cooled erythrocytic cells. The loading of the erythrocytic cells for this aspect of the invention may comprise incubating the erythrocytic cells with an oligosaccharide solution having the oligosaccharide therein and a temperature in a range of temperatures selected from the group consisting of a low phase transition temperature range, an intermediate phase transition temperature range, and a high phase transition temperature range. The lyophilizing is conducted so as to leave a residual water content of equal to or less than about 0.3 gram water per gram dry weight of erythrocytic cells. Preferably, greater than about 80% of the erythrocytic cells survive dehydration and storage. The process of preparing a dehydrated composition may additionally comprise prehydrating the erythrocytic cells, and subsequently hydrating the prehydrated erythrocytic cells.
An additional further embodiment of the present invention comprises a process of preparing loaded erythrocytic cells comprising removing at least a portion of an alcohol (e.g. a sterol) from erythrocytic cells to produce erythrocytic cells having at least three phase transition temperature ranges, and loading (e.g., with an oligosaccharide solution) an oligosaccharide into the erythrocytic cells at a temperature in a range of temperatures approximating one of the three phase transition temperature ranges to produce loaded erythrocytic cells. As previously indicated, the loading may comprise incubating the erythrocytic cells with the oligosaccharide solution at a temperature in a range of temperatures approximating one of the three phase transition temperature ranges.
Additional features of the present invention include a solution for loading erythrocytic cells, an erythrocytic cell composition, and a generally dehydrated composition. The solution for loading erythrocytic cells comprises reduced-alcohol (e.g. reduced-sterol) erythrocytic cells having three phase transition temperature ranges, and an oligosaccharide solution containing the reduced-alcohol erythrocytic cells for loading oligosaccharide from the oligosaccharide solution into the reduced-alcohol erythrocytic cells. External oligosaccharide is taken up via lipid phase endocytosis from the oligosaccharide solution at a temperature in a range of temperatures approximating one of the three phase transition temperature ranges. The erythrocytic cell composition comprises reduced-alcohol erythrocytic cells loaded internally with an oligosaccharide from an oligosaccharide solution. Preferably, the oligosaccharide is loaded from the oligosaccharide solution at a temperature in a range of temperatures selected from the group consisting of a low phase transition temperature range, an intermediate phase transition temperature range, and a high phase transition temperature range. The generally dehydrated composition comprises freeze-dried reduced-alcohol erythrocytic cells effectively loaded internally with at least about 10 mM of the oligosaccharide (e.g., trehalose) therein to preserve biological properties during freeze-drying and rehydration. The amount of the oligosaccharide loaded inside the freeze-dried reduced-alcohol erythrocytic cells may be from about 10 mM to about 200 mM. The freeze-dried reduced-alcohol erythrocytic cells may comprise less than about 0.30 gram of residual water per gram of dry weight erythrocytic cells to increase erythrocytic cell survival upon rehydrating.
The sterol may comprise a steroid alcohol, preferably a steroid alcohol having at least one side chain having 8 to 10 carbon atoms. Preferably further, the sterol may comprise from 25 to 27 carbon atoms. More preferably, the sterol comprises cholesterol, such as cholesterol having the formula: 
The erythrocytic cells preferably comprise erythrocytic membranes respectively including the low phase transition temperature range, the intermediate phase transition, and the high phase transition temperature range. The low phase transition temperature range is greater than about 2xc2x0 C., such as a temperature greater than about 2xc2x0 C. to a temperature equal to or less than about 20xc2x0 C. The intermediate phase transition temperature range is preferably greater than about 20xc2x0 C., such as a temperature greater than about 20xc2x0 C. to a temperature equal to or less than about 30xc2x0 C. The high phase transition temperature range is preferably greater than about 30xc2x0 C., such as a temperature greater than about 30xc2x0 C. to a temperature equal to or less than about 50xc2x0 C., more preferably from about 30xc2x0 C. to about 40xc2x0 C., or from about 32xc2x0 C. to about 38xc2x0 C.
These provisions together with the various ancillary provisions and features which will become apparent to those skilled in the art as the following description proceeds, are attained by the processes, platelets, eukaryotic cells, and erythrocytic cells of the present invention, preferred embodiments thereof being shown with reference to the accompanying drawings, by way of example only, wherein: